Although gender disparities in cardiac disease are recognized, the mechanisms through which pre-menopausal females are protected have not been fully elucidated. Cardiac disease incidence in females increases post-menopause, suggesting a role for estrogen in pre-menopausal cardioprotection. However, clinical trials found no beneficial cardiovascular outcomes from hormone replacement therapy, indicating a better mechanistic understanding is needed. Thus the goal of this study is to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. Classical estrogen-induced transcription regulation is mediated by nuclear estrogen receptors (ER) ER-alpha and ER-beta. However, ER-alpha and ER-beta are also localized to the plasma membrane and can elicit effects through kinase signaling, leading to an increase in S-nitrosylation (SNO), a post-translational modification associated with cardioprotection. We hypothesized that estrogen-related cardioprotection is at least partially mediated by non-nuclear ER signaling leading to an increase in SNO. We tested this using an estrogen-dendrimer conjugate (EDC), which has been demonstrated in mice to be a non-nuclear selective ER modulator (SERM) that does not promote uterine or breast cancer growth. We treated ovariectomized C57BL/6J mice with EDC, dendrimer control, 17-beta-estradiol, or vehicle for two weeks. Isolated hearts were perfused in the Langendorff model and subjected to 30 minutes ischemia and 90 minutes reperfusion. As previously reported, estradiol-treated hearts had decreased infarct size (40.4 2.5% vs. 62.9 5.8%) and increased functional recovery (44.7 4.0% vs. 27.0 2.7%) compared to vehicle-treated hearts. Similar to estradiol, EDC decreased infarct size (40.9 3.6% vs. 63.8 4.7% total ventricle) and improved functional recovery (48.8 3.0% vs. 28.6 2.5%) compared to dendrimer control. Similar protection was seen when mice were treated with EDC for five days (42.1 4.7% vs. 63.8 6.4% infarct and 38.9 2.9% vs. 22.8 2.4% functional recovery for EDC vs. dendrimer). 2-D Difference Gel Electrophoresis showed an increase in protein SNO from hearts treated with EDC for 5 days and 2 weeks compared to dendrimer treatment. Many of the identified proteins have increased SNO in other models of cardioprotection. These results indicate that EDC is as effective as estradiol in providing cardioprotection during ischemia-reperfusion injury in mice, possibly due to increased protein SNO. They further suggest that non-nuclear ER actions play a major role in the protection afforded by estrogen. Thus, EDC and non-nuclear ER signaling pathways could be utilized clinically to provide cardiovascular benefit without promoting cancer cell growth. It is thought that altered gene expression contributes to female cardioprotection. MicroRNAs (miRNAs) are small, non-coding RNA that inhibit gene expression through binding to mRNA. We hypothesize that sex-specific expression patterns of miRNA contribute to differences in expression of cardioprotective proteins. miRNA levels were determined with the Affymetrix GeneChip miRNA array using miRNA from the hearts of male or female mice. Expression of five miRNAs was significantly different between males and females. Because of its role in indirect regulation of eNOS expression, we chose to further study miR-222. Decreased miR-222 in females was confirmed by qRT-PCR and this corresponded with increased eNOS mRNA. The transcription factor, ets-1, was identified as a putative direct target of miR-222 using TargetScan, and increased ets-1 mRNA and protein were confirmed in females. Inhibition of ets-1 expression by miR-222 was established by transfecting a luciferase reporter containing the putative ets-1 3UTR in HEK293 cells treated with either a miR-222 inhibitor or mimic. Furthermore, we confirmed modulation of eNOS levels by a miR-222 inhibitor or mimic in adult rat cardiomyocytes. Thus, inherent sex-specific differences in miRNA expression, and subsequent changes in the expression of cardioprotective proteins, may contribute to female cardioprotection.